The present invention relates to novel compounds able to chelate paramagnetic metal ions and to the use thereof as contrast agents in the technique known as xe2x80x9cMagnetic Resonance Imagingxe2x80x9d (M.R.I.). In particular, the present invention relates to compounds resulting from the conjugation of a carrier consisting of a bile acid residue with molecules endowed with a chelating capacity, as well as their complex chelates with bi- and trivalent paramagnetic metal ions, the salts thereof and the use thereof as contrast agents for M.R.I.
Patent literature reports a number of patents and patent applications concerning chelates of straight and cyclic polyaminopolycarboxylic ligands with paramagnetic metals as contrast agents for M.R.I. Some of these compounds are already in clinical use (Gd-DTPA, N-methylglucamine salt of the gadolinium complex with diethylenetriaminopentaacetic acid, MAGNEVIST(copyright), Schering; Gd-DOTA, N-methylglucamine salt of the gadolinium/1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex, DOTAREM(copyright), Guerbet; Gd-HPDO3A, gadolinium complex with 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, PROHANCE(copyright), BRACCO). These contrast agents are designed for a wholly general use, as they are mainly distributed in the extracellular spaces after administration.
On the contrary, a contrast agent capable of providing diagnostically useful information even in case of small lesions such as liver tissue metastasis, should selectively accumulate in the intracellular spaces of the parenchyma of said organ. Intracellular hepatobiliary agents are capable of entering hepatocytes through the sinusoid membrane and are mainly excreted through the bile. The lipophilic nature of their chelating units is considered responsible for the preferential uptake by hepatocytes (Lauffer, R. B.; Chem. Rev. 87, 901-927, 1987; Lauffer, R. B. et al.; Magn. Reson. Med. 4, 582-590, 1987).
Cholic acid is one of the bile acids resulting from cholesterol catabolism; it is excreted by hepatocytes into bile (Elliot, W. H.; Sterols and bile acids; H. Danielsson and J. Sjxc3x6vall, Eds.; pp 231-278, Elsevier, N.Y., 1985).
M.R.I. contrast agents comprising a residue of a chelating agent conjugated by a spacing chain to a bile acid residue are disclosed in WO95/01958. Said agents are stated to be particularly useful in the imaging of liver and bile ducts.
It has also been found that the bile acid residue makes the chelanting agents including it capable of interacting with plasma proteins thus forming non-covalent bonds with them. Therefore the contrast agents comprising a bile acid residue are also particularly useful in the N.M.R. imaging of the vasal system, as disclosed in Italian patent application MI98A002802, which is herein incorporated by reference in its entirety.
EP 279,307 (Abbott) discloses other polyaminopolycarboxylic chelants, capable of complexing metal ions, conjugated with different substrates, inter alia bile acids, as contrast agents. The only compound disclosed in this patent application is a 111In complex of a conjugate in which a functionalized derivative of EDTA is linked, through an amido bond, to the carboxylic function of a cholic acid. The possibility of chelating paramagnetic metal ions for use in M.R.I. is not referred to in any way.
Betebenner David A. et al. in Bioconjugate Chem. 2; 117-123, 1991 disclose the preparation and characterization of an EDTA derivative conjugated with a cholic acid and of the corresponding chelated complex with 111In. The conjugation takes place through the terminal amino group of a functional derivative of EDTA with a cholic acid ester, by means of a reaction already used for linking chelating units to proteins (Westerberg, D. A. et al., J. Med. Chem. 32, 236-243, 1989; Brechbiel, M. W. Et al., Inorg. Chem. 25, 2772-2781, 1986). Biodistribution and scintigraphic imaging studies carried out with this compound show that, after uptake by the liver, it rapidly passes into the intestine.
All known agents comprising the residue of a bile acid are characterized by the presence of a single chelating unit and, therefore, of a single metal ion coordinated per each molecule of the agent itself.
The present invention relates to compounds capable of chelating at least two metal ions. Said compounds are obtained by conjugation of a carrier consisting of a bile acid residue with two chelating molecules, said conjugation taking place through a central reactive polyfunctional substrate or central synton. In particular, the bonds between the central syntons of the invention and the chelating units are obtained by means of functional residues suitably selected so that said chelating units can keep unaltered their capability to coordinate the metal ion and the stability of the resulting chelates does not decrease.
More particularly, the present invention relates to compounds of general formula (I) 
wherein:
A is a polyfunctional reactive substrate containing at least three functional groups deriving from any polyvalent organic residue which can be aliphatic with open chain, optionally branched, or alicyclic, or heterocyclic containing N, O and/or S, or aromatic or heteroaromatic;
Z is a bile acid residue;
Y1 and Y2, which can be the same or different, are the residue of a chelating agent of the bi-trivalent metal ions having atomic numbers ranging between 20 and 31, 39, 42, 43, 44, 49, and between 57 and 83;
L1, L2 and L3, which can be the same or different, are a single bond between the functional groups Y1and A, Y2 and A and/or Z and A, or a spacer comprising at most 20 carbon atoms.
Bile acid herein means all bile acids obtained by bioconversion or synthetic modification of cholesterol, particularly cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, lithocholic acids and the derivatives thereof, including those with taurine and glycine. Said residues are linked to the L3 group through covalent bonds involving the hydroxy group, optionally converted to an amino group, at the 3-position or the carboxy group at the 24-position of the cholane skeleton.
Preferred compounds are those in which L1 and L2, which can be the same or different, are a spacer chain of formula (II) 
wherein:
Q is a C1-C8 alkyl chain optionally substituted with 1 to 3 OH groups;
s is an integer 0 to 5;
R1 is an H atom, or a C1-C5 alkyl group;
X is 
L3 is a group of formula (III) 
in which: X and s have the above defined meanings (with the proviso that when s is different from 0 then CO and X are present).
Particularly preferred are the compounds in which L1 and L2 are selected from those of formula (IV), (V), (VI) 
L3 is a single bond between the Z and A functional groups or a group 
in which:
n is an integer 1 to 8, and
R1 has the above defined meanings.
The invention also relates to the complex chelates of said compounds of formula (I) with the bi- and trivalent ions of the metal elements having atomic numbers ranging between 20 and 31, 39, 42, 43, 44, 49, and between 57 and 83, as well as the salts thereof with physiologically compatible organic bases selected from primary, secondary, tertiary amines or basic amino acids, or with inorganic bases the cations of which are sodium, potassium, magnesium, calcium or mixtures thereof, or with anions of physiologically acceptable organic acids selected, for example, from acetate, succinate, citrate, fumarate, maleate, oxalate, or with anions of inorganic acids such as ions of halo acids, i.e. chlorides, bromides, iodides.
The invention also relates to the preparation of the compounds of general formula (I) as well as the complex salts thereof and the use thereof for the preparation of pharmaceutical formulations for the diagnostic use, in particular for M.R.I. contrast formulations.
Furthermore, the compounds of the invention can optionally be chemically conjugated with suitable macromolecules or englobated in suitable carriers.
Preferred compounds are those in which Z is a residue deriving from the following bile acids or derivatives thereof with taurine and glycine: 
A is a polyfunctional reactive substrate having preferably amino and/or carboxy groups. Preferred examples of substrates A are reported in Table 1.
The compounds 1-17 can be used as such or as amino- or carboxy-protected or activated derivatives.
Y1 and Y2 are preferably the residues of two polyaminopolycarboxylic ligands, either in the form of acids or of derivatives thereof. More particularly, Y1 and Y2 are preferably residues of diethylenetriaminopentaacetic (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic (DOTA), 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic (HPDO3A), 4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic (BOPTA) acids, or of N-[2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl]-N-[2-[bis(carboxymethyl)amino]ethyl]glycine (EOB-DTPA) and N,N-bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]amino]-ethyl]glycine (DTPA-BMA).
Particularly preferred are the compounds in which Z is a cholic acid residue, A is a polyfunctional substrate selected from those of formulae 1, 2, 3, 4 and 17 and Y1 and Y2, which can be the same or different, are selected from residues shown in the following Table 2:
Particularly preferred are the following compounds:
[3xcex1[3(S),4(S)],5xcex2,12xcex1]-3-[[[trans-3,4-bis[[[5-[bis[2-[bis(carboxymethyl)-amino]ethyl]amino]-5-carboxypentyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oic acid;
[3xcex1[3(S),4(S)],5xcex2,12xcex1]-3-[[[trans-3,4-bis[[[2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-2-carboxyethyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oic acid;
(3xcex1,5xcex2,12xcex1)-3-[[[trans-3,4-bis[[[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oic acid;
[3xcex1[3(S),4(S)],5xcex2,12xcex1]-3-[[[trans-3,4-bis[[[5-[[2-[bis(carboxymethyl)-amino]ethyl](carboxymethyl)amino]-5-carboxypentyl]amino]carbonyl]-1-pyrrolidinyl]carbonyl]oxy]-12-hydroxycholan-24-oic acid;
[3xcex1[1(S),2(S)],5xcex2,7xcex112xcex1]-3-[[[[cis-1,2-bis[[[5-[bis[2-[bis(carboxymethyl)-amino]ethyl]amino]-5-carboxypentyl]amino]carbonyl]-4-cyclopentyl]amino]-carbonyl]oxy]-7,12-dihydroxycholan-24-oic acid;
[3xcex1[1(S)],5xcex2,7xcex1,12xcex1]-3-[[[[cis-1-[[[5-[bis[2-[bis(carboxymethyl)amino]ethyl]-amino]-5-carboxypentyl]amino]carbonyl]-2-[[[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethyl]amino]carbonyl]-4-cyclopentyl]amino]carbonyl]oxy]-7,12-dihydroxycholan-24-oic acid;
[3xcex2[3(S),5(S)],5xcex2,7xcex1,12xcex1]-3-[[[3,5-bis[[4-[bis[2-[bis(carboxymethyl)amino]-ethyl]amino]-4-carboxy-1-oxobutyl]amino]phenyl]carbonyl]amino]-7,12-dihydroxy-cholan-24-oic acid;
as well as the chelates and the physiologically compatible salts thereof.
Suitable ions for forming complex salts with the chelating agents of general formula (I) are bivalent or trivalent ions of the elements having atomic numbers ranging between 20 and 31, 39, 42, 43, 44, 49, between 57 and 83; particularly preferred are Fe(2+), Fe(3+), Cu(2+), Cr(3+), Gd(3+), Eu(3+), Dy(3+), La(3+), Yb(3+), or Mn(2+), or also radioisotopes such as 51Cr, 67Ga, 68Ga, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi.
The compounds of the present invention have shown good tolerability as well as water solubility.
The improvement of the M.R.I. imaging that the radiologist can observe, namely an increase in the contrast between healthy and affected tissues, is certainly an aid for diagnosis. Said improvement is generally obtained by administering the patient with suitable exogenous compounds, such as the suitably complexed paramagnetic metal ions, capable of significantly changing the relaxivity of the water protons of the tissue they are in contact with, when said protons are subjected to an outer magnetic field.
The change induced by the presence of two paramagnetic metal ions per molecule of the administered agent, characteristic of the compounds of the invention, is significantly enhanced thereby obtaining a contrast between healthy and affected tissue which is already diagnostically useful at lower dosages of contrast agents.
An advantage of the complex chelates of the invention is therefore that they can be used in low-dosage diagnostic formulations capable of providing good-quality images while remarkably decreasing the toxicity and the discomfort unavoidably involved by the administration of an exogenous substance, such as a contrast agent.
The compounds of the invention can therefore be advantageously administered at doses ranging from 0.005 to 1.0 mmol/kg, and preferably from 0.01 to 0.1 mmol/kg.
Thanks to the presence in the molecule of a carrier such as the bile acid residue, the agents of the invention can interact with plasma proteins and form non-covalent bonds therewith. This property thus provides high relaxivity in the serum and such long permanency in blood as to make them particularly suitable for M.R.I. imaging of the vascular system.
It has surprisingly found that, in addition to increasing the number of the paramagnetic metal ions present, which involves advantages in terms of relaxivity and signal intensity, the introduction of a second chelating unit in the compounds of the invention induces a remarkable change in the excretion mechanism thereof. The compounds of the invention, in fact, are surprisingly almost completely excreted thorough the renal route, by glomerular filtration. The experimental results of the pharmacokinetic screening in rats indicate that during a period of 0 to 480 min, 36.2% of the administered compound was eliminated with urine and only 0.175% of the injected dose with bile. A detailed description of the experimental conditions as well as of the obtained results is included, as a non-limiting example, in the experimental section.
The compounds of the invention, therefore, while maintaining unaffected the properties deriving from the presence of the bile acid residue, do not enter liver intracellular spaces and are excreted through the bile only to a very limited extent, contrary to the compounds of the prior art containing one chelant residue only.
This involves, on the one hand, an unexpected improvement in terms of toxicity and, on the other hand, total absence of metabolization by hepatocytes.
From still another point of view, the agents of the invention have remarkably increased half-life in blood, as they are captured and englobated by the hepatocytes only to a very low extent. This further unexpected property, as well as the increased relaxivity in serum, contribute to make the agents of the present invention particularly suitable for the imaging of the vascular system in general, and particularly of coronaries, even at the lows dosages used.
The compounds of general formula (I) can be prepared with synthetic methods usually employed in industrial technique.
In particular, the preparation of compounds in which the two chelating units are the same is performed according to a synthetic process similar to that illustrated in the following Scheme, representing the synthesis of the compound of Example 1, as detailed in the experimental section. 
Briefly, the synthetic process of the scheme comprises the following steps:
1) synthesis of the central polyfunctional residue A in a suitable form, in which the functional groups can independently be suitably protected or activated;
2) synthesis of the suitably functionalized chelant residue B, i.e. of a chelating agent capable of stably coordinating the metal ions as well as of covalently binding to the central polyfunctional residue through a suitable functional group;
3) linking between central polyfunctional residue and chelant residues and isolation of intermediate C;
4) synthesis of a suitably functionalized bile acid D capable of stably binding to the central polyfunctional residue through a suitable functional group;
5) optional cleavage and/or activation of the third functional residue A, linking between the resulting compound and D and isolation of E;
6) cleavage of any protective groups and isolation of polyacid F;
7) complexation of the metal ions and isolation of the chelated complex G.
When the two chelating units of the compounds are different, the contrast agents of the invention can be prepared by using a similar synthetic process in which intermediate C is prepared by reacting the suitable polyfunctional residue A with a first chelant residue B1, subsequent activation of a second functional group A and linking between it and the second chelant residue B2.
Briefly, said process comprises the following synthetic steps:
1) synthesis of the central polyfunctional residue A in a suitable form, in which the functional groups can independently be suitably protected or activated;
2) synthesis of the two suitably functionalized chelant residues B1 and B2, i.e. of two chelant units capable of stably coordinating the metal ions as well as of covalently binding to the central polyfunctional residue through a suitable functional group;
3) linking between polyfunctional residue and a first chelant residue B1; optional cleavage and/or activation of a second functional residue A, linking between it and a chelating unit B2 and isolation of intermediate C;
4) synthesis of a suitably functionalized bile acid D, capable of stably binding to the central polyfunctional residue through a suitable functional group;
5) optional cleavage and/or activation of the third functional residue A, linking between the resulting compound and D and isolation of E;
6) cleavage of any protective groups and isolation of polyacid F;
7) complexation of the metal ions and isolation of the chelated complex G.
Said compounds, or the suitable precursors and/or functional derivatives thereof, can be easily obtained according to the known synthetic techniques.
By way of example, compound 2 is commercially available, derivatives of compound 7 in which the amino groups are suitably protected can be prepared as described in J. Am. Chem. Soc. 1996, 118, 2567 or in Polymer 1982, 23, 771; compound 5 as described in Tetrahedron Lett. 1993, 34, 4989; compound 4 dimethyl ester as described in J. Org. Chem. 1989, 54, 5115; compound 1 dimethyl ester with the amino residue suitably protected as benzyl derivative, as described in Bull. Soc. Chim. Fr. 1988, 579; compound 3 derivative in which the amino functions are appropriately protected, as described in Chem. Commun. 1998, 1629; compounds 6 and 8 dimethyl esters in which the amino functions are suitably protected, as described in J. Chem. Soc. Perkin trans. I 1991, 409 and Tetrahedron Lett. 1994, 35, 4515 respectively; compound 13 dimethyl ester as described in J. Med. Chem. 1990, 33, 1561; anhydride 17 as described in Chem. Pharm. Bull. 1984, 32, 805.
The residues of Table 2 can be prepared using known techniques. The residues a, b and c can, for example, be prepared analogously to what described in Bioconjugate Chem. 1999, 10, 137; the preparation of the residue d is described in WO 9601655, that of the residue e in WO 9528967, that of f is described in the experimental section, Example 4.
Preferred bile acid functional derivatives for the preparation of the compounds of the invention are, for example, the 3xcex2-amino derivatives thereof, obtained starting from corresponding 3xcex1-hydroxyls following the synthetic process described in Synth. Commun. 1998, 28, 109.
Also preferred are chloroorthoformates obtained from the corresponding 3xcex1-hydroxyls according to the synthetic process described in J. Am. Chem. Soc. 1997, 117, 640.
Both cited derivatives can be used in the linking reactions directly, or they can be further modified by reacting the respective functional groups with suitable bifunctional spacers.
A list of particularly preferred bile acid functional derivatives is reported hereinbelow. For many compounds, the list also reports a bibliographic reference concerning the preparation thereof.
Bile Acid Functional Derivatives 
The linking between polyfunctional central nucleus and chelant residue can take place, for instance, between a suitably functionalized amino group present on the chelant and a carboxylic residue present on the polyfunctional central residue or, vice versa, between a carboxylic residue of the suitable ligand functional derivative and an amino residue of the central polyfunctional residue to form an amide in both cases.
The linking between the central polyfunctional residue and the bile acid residue can take place by formation of an amido bond, for example between the acid group at the 24-position of the bile residue and an amino group present in the central polyfunctional synton, according to a procedure widely described and used in the preparation of, for example, peptides (Tetrahedron, 32, 2211, 1976).
Alternatively, the amido bond can be formed between the amino group of a bile acid 3xcex2-amino derivative and a carboxylic group of the central polyfunctional residue, or between the chlorocarbonyl residue of the bile acid chloroformate and an amino group of the central polyfunctional residue.
Said linking may optionally be preceded by cleavage and/or activation of the functional group of the central unit involved in the reaction.
Amino functional groups are usually protected by transformation into the corresponding benzyl, carbobenzyloxy (Cbz) or tert-butyloxycarbonyl (BOC) derivatives. In the first two cases, for example, the cleavage reaction can take place by hydrogenation of the benzyl or Cbz derivatives, in the presence of a suitable metal catalyst, for example Pd/C. In the third case, cleavage can be obtained in acidic conditions, for example with CF3COOH.
The carboxylic residues are usually protected by transformation into suitable esters. The subsequent deprotection of carboxylic functions of the two chelant units can be obtained by hydrolysis of the protective ester groups, for example with LiOH, CF3COOH or iodotrimethylsilane in a suitable organic solvent, such as dioxane, CH2Cl2 or CH3CN or an alcohol, such as i.PrOH.
The compounds of the invention have a wide range of applications, since they can be administered through the intravasal (for instance intravenous, intraarterial, intracoronaric, intraventricular routes, and so on), intrathecal, intraperitoneal, intralymphatic and intracavital routes. The compounds are also suitable for the oral or enteral administration, and therefore, for the imaging of the gastrointestinal tract.
For the parenteral administration, the compounds of the invention are preferably formulated as sterile solution or aqueous suspension, with pH preferably ranging from 6.0 to 8.5. Said aqueous solutions or suspensions can be administered in concentrations ranging from 0.002 to 1.0 molar. The resulting formulations can be lyophilized and supplied as such, to be reconstituted prior to use.
For the gastrointestinal use or for injection to body cavities, these agents can be formulated as a solution or suspension containing suitable additives in order to, for example, control viscosity.
For the oral administration they can be formulated according to preparation methods routinely used in the pharmaceutical technique or as coated formulations to gain extra protection from the acid pH of stomach, inhibiting the release of the chelated metal ion, which usually occurs at typical pH values of gastric juices.
Other excipients, such as sweeteners and/or flavoring agents, can be also added according to known techniques of pharmaceutical formulation.
The solutions or suspensions of the compounds of this invention can also be formulated as aerosol to be used in aerosol-bronchography and instillation.
As far as diagnostic imaging is concerned, the chelates of this invention can also be used as radiopharmaceuticals in nuclear medicine both in the diagnostic and therapeutic field. However, in this case the metal ion which is chelated is a radioisotope, such as 51Cr, 67Ga, 68Ga, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi.
Preferred cations of inorganic bases which can be suitably used to salify complex chelates of this invention particularly comprise ions of alkali or alkaline-earth metals such as potassium, sodium, calcium, magnesium and the mixtures thereof.
Preferred cations of organic bases suitable for the above mentioned aim, comprise, among others, those of primary, secondary and tertiary amines such as ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N,N-dimethylglucamine.
Preferred anions of inorganic acids which can be suitably used for the salification of complex chelates of this invention particularly comprise anions of the hydrohalo acids such as chlorides, bromides, iodides or other anions such as sulfate.
Preferred anions of organic acids suitable for the above mentioned aim comprise those of acids routinely used in pharmaceutical technique for the salification of basic substances, such as acetate, succinate, citrate, maleate and fumarate.
Preferred cations and anions of amino acids comprise, for example, those of taurine, glycine, lysine, arginine, ornithine or of aspartic and glutamic acids.
The compounds of the invention can be encapsulated or in liposomes or form the constituents of their chemical structure and used as uni- or multilamellar vesicles.
The compounds of the invention can also be conjugated with macromolecules or englobated into or combined with suitable carriers.
In the following, a non-limiting list of preferred compounds of the invention is reported.






